Dietary supplementation with rumen-protected capsicum during the transition period improves the metabolic status of dairy cows.

In ruminants, it has been observed that capsicum oleoresin can alter insulin responses and that high-intensity artificial sweetener can increase glucose absorption from the small intestine. Because glucose metabolism and insulin responses are critical during early lactation, these supplements might have an effect on the metabolic status of dairy cows during the transition period. The objective of this experiment was to evaluate the effects of rumen-protected capsicum oleoresin fed alone or in combination with artificial sweetener during the transition period on lactational performance and susceptibility to subclinical ketosis in dairy cows. Fifteen primiparous and 30 multiparous Holstein cows (a total of 39 cows finished the study) were arranged in a randomized complete block design during d -21 to 60 relative to parturition. Cows within block were randomly assigned to one of the following treatments: no supplement (CON), supplementation with 100 mg of rumen-protected capsicum/cow per day (RPCap), or RPCap plus 2 g of high-intensity artificial sweetener/cow per day (RPCapS). For both the RPCap and RPCapS treatments, only rumen-protected capsicum was fed during the dry period. From d 8 to 11 of lactation, intake was limited to 70% of predicted dry matter intake to induce subclinical ketosis. Production variables were recorded daily, samples for milk composition were collected on wk 2, 4, 6, and 8, and blood samples were collected on wk -2, 1, 2, and 4 of the experiment for analysis of metabolic hormones and blood cell counts. Supplementation with rumen-protected capsicum increased serum insulin and decreased ß-hydroxybutyrate concentrations precalving, indicating a decrease in lipolysis. During the lactation period, RPCap was associated with a trend for increased milk production and feed efficiency following the ketosis challenge. Supplementation with RPCapS appeared to negate the response to rumen-protected capsicum. All cows developed subclinical ketosis during the challenge, and this was not affected by treatment. We conclude that treatments did not decrease susceptibility to subclinical ketosis; however, dietary supplementation with rumen-protected capsicum was effective at improving energy status precalving and tended to increased milk production and feed efficiency. The mechanism underlying these responses is unclear.

Acceptance of flavored concentrate premixes by young ruminants following a short-term exposure.

Three cafeteria feeding design experiments were conducted to test whether young ruminants have flavor preferences. Experiment 1 was with 11 Dorset × Suffolk weaned lambs of both sexes, aged 5 mo and averaging 47.5 (standard deviation = 5.8) kg of body weight. The lambs were offered a choice of 5 flavored concentrate premixes (FCP) and an unflavored control for 5 min 4 times over 10 d. The FCP were prepared by mixing 200 to 300 g/t (as-is basis) of synthetic flavors (vanilla, milky, spicy/fenugreek, red summer fruits, and molasses) into a basal diet. The unflavored control and the milky flavor were consumed in greater amounts than all other flavors at 83.9 and 65.8 g/test, respectively. The consumption rate of FCP (g/min) was similar among treatments. Lambs spent more time consuming the milky flavor and the control at 123 and 144 s/test, respectively, compared with all other FCP (average of 65 s/test). In experiment 2, 12 weaned female Holstein Friesian calves (56-68 d of age) averaging 75.8 kg (standard deviation = 8.45) of body weight were offered a choice of 4 FCP (vanilla, milky, spicy/fenugreek, and red summer fruits) at an inclusion rate of 150 to 200 g/t (as-is basis) and the unflavored control for 5 min 4 times over 10 d. The average consumption rate was 27.8 g/min, and there were no differences among FCP. In experiment 3, a choice of 4 FCP with 2 different flavor combinations (vanilla-fenugreek and milky-vanilla) included at 75 g/t (as-is basis; low) or 150 g/t (high) was offered to a total of 12 weaned female Holstein Friesian calves (47-62 d of age) with an average body weight of 65.3 kg (standard deviation = 7.91). The FCP were offered daily for 14 d for 30 to 60 min/d. Vanilla-fenugreek (low) was consumed less at 57.5 g/test per calf compared with the other FCP (average of 87.5 g/test per calf). There were no other differences among FCP in experiment 3. Overall, compared with the control, flavors used in the present experiments did not affect feed intake of weaned lambs and calves. Other factors, such as taste, sight, texture of the feed, effect of the dams as previous experience (via maternal ingestion, which influences neonatal feeding), and their interactions, may also play a role in flavor preferences of young ruminants.

Effects of phytonutrients alone or in combination with monensin on productivity in lactating dairy cows.

This experiment was conducted to investigate the effects of phytonutrients, compared with monensin as a positive control, on productivity, milk fatty acids, fat mobilization, and blood cells in lactating dairy cows. Thirty-six Holstein cows were used in a 9-wk randomized complete block design study. Following a 2-wk covariate period, cows were blocked by days in milk, parity, and milk yield and randomly assigned to 1 of 3 treatments (12 cows/treatment): 450 mg/cow per day of monensin (MO), 250 mg/cow per day of capsicum plus 450 mg/cow per day of MO (MOCAP), and 1,000 mg/cow per day of a mixture of cinnamaldehyde, eugenol, and capsicum (CEC). Dry matter intake and milk yield were not affected by treatment. Supplementation of CEC increased feed efficiency compared with MO, but did not affect feed efficiency on an energy-corrected milk basis. Milk composition (fat, protein, and lactose), milk fatty acid profile, and blood concentrations of nonesterified fatty acids and ß-hydroxybutyrate were also not affected by treatment. The expression of hormone-sensitive lipase in adipose tissues tended to increase for MOCAP compared with MO. Counts of total white blood cell, neutrophils, lymphocytes, eosinophils, and basophils were not affected by treatment, although monocytes count tended to be decreased by CEC. Treatments had no effect on red blood cells, hemoglobin, and platelets. Results indicate that dietary supplementation of CEC and capsicum had no production or other effects in dairy cows, compared with MO, except CEC increased feed efficiency and tended to decrease blood monocytes count.

Host-mediated effects of phytonutrients in ruminants: A review.

Plants produce an extensive array of organic compounds derived from secondary metabolism that may be useful in animal nutrition because of their chemical makeup. These plant-derived bioactive compounds, also referred to as phytonutrients (PN) or phytobiotics, have been shown to express antimicrobial activities against a wide range of bacteria, yeast, and fungi and have been investigated as rumen modifiers in ruminant nutrition. Studies have reported that PN may inhibit deamination of AA and methanogenesis in the rumen and shift fermentation toward propionate and butyrate. Most of the experiments, however, have been conducted in vitro, and responses have been highly variable and inconsistent in animal experiments. In addition, some studies have reported that PN had positive effects on productivity, although rumen fermentation was not affected. Other than antimicrobial effects in the gut, PN are known to bind specific receptors expressed in neurons, intestines, and other cells and exhibit related physiological effects in nonruminants. The receptor-mediated effects include immune responses, oxidative stress, and insulin secretion and activity. Some PN, due to their phenolic nature, are likely less susceptible to microbial degradation in the rumen and may exhibit activities postruminally, similar to their mode of action in nonruminant species. This opens a new area of research in ruminants, including effects of PN on the animal's immune system, postruminal nutrient use, and animal physiology. Although limited, studies with ruminants provide first evidence of PN's regulatory effects on the host responses. For example, PN were reported to regulate immune cells related to adaptive and innate immunity in challenged or nonchallenged dairy cows. Supplementation of PN reduced oxidative stress by decreasing lipid peroxidation and increasing endogenous antioxidants in ruminants. Additionally, insulin secretion and sensitivity were reportedly regulated by PN in dairy cows. The regulatory effects of PN on immunity may be beneficial for immune suppression and inflammation in dairy cows. In addition, PN could positively affect energy partitioning for milk production through their effects on insulin secretion and sensitivity. Further research is needed to elucidate the effect and mode of action of PN on immune function and animal energetics.

Effects of rumen-protected Capsicum oleoresin on productivity and responses to a glucose tolerance test in lactating dairy cows.

The objective of this experiment was to investigate the effects of rumen-protected Capsicum oleoresin (RPC) supplementation on feed intake, milk yield and composition, nutrient utilization, fecal microbial ecology, and responses to a glucose tolerance test in lactating dairy cows. Nine multiparous Holstein cows were used in a replicated 3 × 3 Latin square design balanced for residual effects with three 28-d periods. Each period consisted of 14 d for adaptation and 14 d for data collection and sampling. Treatments were 0 (control), 100, and 200 mg of RPC/cow per day. They were mixed with a small portion of the total mixed ration and top-dressed. Glucose tolerance test was conducted once during each experimental period by intravenous administration of glucose at a rate of 0.3 g/kg of body weight. Dry matter intake was not affected by RPC. Milk yield tended to increase for RPC treatments compared to the control. Feed efficiency was linearly increased by RPC supplementation. Concentrations of fat, true protein, and lactose in milk were not affected by RPC. Apparent total-tract digestibility of dry matter, organic matter, and crude protein was linearly increased, and fecal nitrogen excretion was linearly decreased by RPC supplementation. Rumen-protected Capsicum oleoresin did not affect the composition of fecal bacteria. Glucose concentration in serum was not affected by RPC supplementation post glucose challenge. However, compared to the control, RPC decreased serum insulin concentration at 5, 10, and 40 min post glucose challenge. The area under the insulin concentration curve was also decreased 25% by RPC. Concentration of nonesterified fatty acids and ß-hydroxybutyrate in serum were not affected by RPC following glucose administration. In this study, RPC tended to increase milk production and increased feed efficiency in dairy cows. In addition, RPC decreased serum insulin concentration during the glucose tolerance test, but glucose concentration was not affected by treatment.

Reducing gut effects from Cryptosporidium parvum infection in dairy calves through prophylactic glucagon-like peptide 2 therapy or feeding of an artificial sweetener.

Glucagon-like peptide 2 (GLP-2) therapy was shown previously to reduce inflammation-related gut damage from coccidiosis in dairy calves, and feeding of artificial sweetener stimulates GLP-2 secretion from intestinal L cells. The purpose of this study was to determine whether GLP-2 treatment or artificial sweetener feeding beginning 1 wk before an experimental inoculation with the coccidian parasite Cryptosporidium parvum can reduce infection-related intestinal damage in Holstein bull calves. Newborn calves were assigned to 1 of 4 treatment groups of 6 calves each, including noninfected control calves injected s.c. every 12 h with control buffer (CON), infected control calves injected s.c. every 12 h with control buffer (INF), infected calves injected s.c. every 12 h with 50 µg/kg of body weight of GLP-2 (GLP2), and infected calves injected s.c. every 12 h with control buffer and supplemented in the diet with Sucram (Pancosma, Geneva, Switzerland) at 400 mg/kg of dry matter of milk replacer (SUC). Treatments were initiated on d 1, and calves in INF, GLP2, and SUC were orally dosed on d 8 with 12,500 C. parvum oocysts. Fecal scores were recorded daily, plasma was collected on d 1, 8, 12, 15, and 18 to evaluate markers of inflammation, and fecal samples were collected on d 1, 8, and every other day thereafter to determine the presence of oocysts. Calves were euthanized on d 18 for collection of intestinal tissues and histological and gene expression analyses. Relative to CON, calves in INF exhibited an increase in diarrhea severity, increased plasma serum amyloid A concentration on d 15 and 18, reduced intestinal villus height, increased villus apoptosis and crypt cell proliferation, and increased intestinal mRNA expression of MARVELD2 and GPX2. However, calves in SUC and GLP2 had reduced diarrhea severity and fecal C. parvum oocyst shedding, reduced plasma serum amyloid A concentration on d 15 and 18, and, depending on the intestinal segment, increased villus height, reduced crypt cell proliferation, and reduced mRNA expression of MARVELD2, GPX2, and other tight junction proteins relative to INF. Lastly, GLP2 and SUC exhibited increased intestinal mass-to-length ratio and decreased length-to-empty body weight ratio relative to INF. Our findings suggest that GLP-2 and Sucram treatments administered before a low-level C. parvum exposure may contribute to fewer effects on intestinal integrity, morphology, and inflammation in response to infection, and shorter, denser intestines.

Effects of rumen-protected Capsicum oleoresin on immune responses in dairy cows intravenously challenged with lipopolysaccharide.

The objective of this experiment was to investigate the effects of rumen-protected Capsicum oleoresin (RPC) on productivity and immune responses including feed intake, milk yield and composition, white and red blood cells, lipid peroxidation, and blood concentration of cortisol, haptoglobin, glucose, and insulin in lactating dairy cows experimentally challenged with lipopolysaccharide (LPS). The experiment was a replicated 3 × 3 Latin square design with 9 multiparous Holstein cows in three 28-d periods. Treatments were 0 (control), 100, and 200 mg of RPC/cow per day, mixed with small portions of the total mixed ration and top-dressed. Bacterial LPS was intravenously administered at 1.0 µg/kg of body weight in the last week of each experimental period, and blood samples were collected at 0, 2, 4, 8, and 24 h after administration. Dry matter intake, milk yield, and white blood cells including neutrophils, lymphocytes, monocytes, and eosinophils were decreased, and rectal temperature, hemoglobin, and serum concentrations of cortisol and haptoglobin were increased by LPS. Red blood cells, platelets, and plasma concentration of thiobarbituric acid reactive substances were not affected by LPS. Dry matter intake, milk yield, and milk composition in the 5 d post-LPS challenge were not affected by RPC. Rectal temperature, white blood cells, red blood cells, hemoglobin, and platelets were also not affected by RPC. Compared with the control, RPC tended to decrease cortisol at 2 h following LPS challenge and decreased haptoglobin concentration in serum across sampling points. Concentration of thiobarbituric acid reactive substances in plasma was decreased by RPC at 24 h post-LPS challenge. Glucose and insulin were not affected by RPC, but serum insulin concentration at 8 h was lowered by RPC compared to the control. Collectively, RPC had no or subtle effects on feed intake, milk yield and composition, rectal temperature, white and red blood cells, and serum glucose and insulin concentration in dairy cows challenged by LPS. However, RPC tended to decrease cortisol and decreased concentrations of haptoglobin and thiobarbituric acid reactive substances in blood following LPS challenge. Data suggest that dietary supplementation of RPC may modulate acute phase responses induced by bacterial infection in lactating dairy cows.

Glucagon-like peptide 2 and its beneficial effects on gut function and health in production animals.

Numerous endocrine cell subtypes exist within the intestinal mucosa and produce peptides contributing to the regulation of critical physiological processes including appetite, energy metabolism, gut function, and gut health. The mechanisms of action and the extent of the physiological effects of these enteric peptides are only beginning to be uncovered. One peptide in particular, glucagon-like peptide 2 (GLP-2) produced by enteroendocrine L cells, has been fairly well characterized in rodent and swine models in terms of its ability to improve nutrient absorption and healing of the gut after injury. In fact, a long-acting form of GLP-2 recently has been approved for the management and treatment of human conditions like inflammatory bowel disease and short bowel syndrome. However, novel functions of GLP-2 within the gut continue to be demonstrated, including its beneficial effects on intestinal barrier function and reducing intestinal inflammation. As knowledge continues to grow about GLP-2's effects on the gut and its mechanisms of release, the potential to use GLP-2 to improve gut function and health of food animals becomes increasingly more apparent. Thus, the purpose of this review is to summarize: (1) the current understanding of GLP-2's functions and mechanisms of action within the gut; (2) novel applications of GLP-2 (or stimulators of its release) to improve general health and production performance of food animals; and (3) recent findings, using dairy calves as a model, that suggest the therapeutic potential of GLP-2 to reduce the pathogenesis of intestinal protozoan infections.

Effects of dietary Capsicum oleoresin on productivity and immune responses in lactating dairy cows.

This study investigated the effect of Capsicum oleoresin in granular form (CAP) on nutrient digestibility, immune responses, oxidative stress markers, blood chemistry, rumen fermentation, rumen bacterial populations, and productivity of lactating dairy cows. Eight multiparous Holstein cows, including 3 ruminally cannulated, were used in a replicated 4×4 Latin square design experiment. Experimental periods were 25 d in duration, including a 14-d adaptation and an 11-d data collection and sampling period. Treatments included control (no CAP) and daily supplementation of 250, 500, or 1,000 mg of CAP/cow. Dry matter intake was not affected by CAP (average 27.0±0.64 kg/d), but milk yield tended to quadratically increase with CAP supplementation (50.3 to 51.9±0.86 kg/d). Capsicum oleoresin quadratically increased energy-corrected milk yield, but had no effect on milk fat concentration. Rumen fermentation variables, apparent total-tract digestibility of nutrients, and N excretion in feces and urine were not affected by CAP. Blood serum ß-hydroxybutyrate was quadratically increased by CAP, whereas the concentration of nonesterified fatty acids was similar among treatments. Rumen populations of Bacteroidales, Prevotella, and Roseburia decreased and Butyrivibrio increased quadratically with CAP supplementation. T cell phenotypes were not affected by treatment. Mean fluorescence intensity for phagocytic activity of neutrophils tended to be quadratically increased by CAP. Numbers of neutrophils and eosinophils and the ratio of neutrophils to lymphocytes in peripheral blood linearly increased with increasing CAP. Oxidative stress markers were not affected by CAP. Overall, in the conditions of this experiment, CAP did not affect feed intake, rumen fermentation, nutrient digestibility, T cell phenotypes, and oxidative stress markers. However, energy-corrected milk yield was quadratically increased by CAP, possibly as a result of enhanced mobilization of body fat reserves. In addition, CAP increased neutrophil activity and immune cells related to acute phase immune response.

Responses of the mammary transcriptome of dairy cows to altered photoperiod during late gestation.

Cows exposed to short day photoperiod (SD, 8L:16D) during the 60-day nonlactating period prior to parturition produce more milk in their subsequent lactation compared with cows exposed to long day photoperiod (LD, 16L:8D). Although this response is well established in dairy cows, the underlying mechanisms are not understood. We hypothesized that differential gene expression in cows exposed to SD or LD photoperiods during the dry period could be used to identify the functional basis for the subsequent increase in milk production during lactation. Pregnant, multiparous cows were maintained on an SD or LD photoperiod for 60 days prior to parturition. Mammary biopsies were obtained on days -24 and -9 relative to parturition and Affymetrix GeneChip Bovine Genome Arrays were used to quantify gene expression. Sixty-four genes were differentially expressed (P ≤ 0.05 and fold-change ≥ |1.5|) between SD and LD treatments. Many of these genes were associated with cell growth and proliferation, or immune function. Ingenuity Pathway Analysis predicted upstream regulators to include TNF, TGF-ß1, interferon-γ, and several interleukins. In addition, expression of 125 genes was significantly different between day -24 and day -9; those genes were associated with milk component metabolism and immune function. The interaction of photoperiod and time affected 32 genes associated with insulin-like growth factor I signaling. Genes differentially expressed in response to photoperiod were associated with mammary development and immune function consistent with the enhancement of milk yield in the ensuing lactation. Our results provide insight into the mechanisms by which photoperiod affects the mammary gland and subsequently lactation.

Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland.

During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17ß-estradiol (InsHPrlE). The relative expression of ß-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of ß-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation.

Effects of increased milking frequency during early lactation on milk yield and udder health of primiparous Holstein heifers.

In dairy cows, increased milking frequency (IMF) during early lactation stimulates an increase in milk yield that partially persists through the remainder of lactation. However, the effects of IMF on lactation performance and udder health of primiparous heifers have not been clearly established. The objective of this study was to determine the effects of IMF during early lactation on milk yield and composition, udder edema, and teat-end condition of primiparous Holstein heifers. Thirteen heifers were assigned at parturition to unilateral frequent milking (twice-daily milking of the left udder half (2X), and 4-times-daily milking of the right udder half (4X)) from d 1 to 21 of lactation. Heifers were milked twice daily at 0130 and 1330 h, with additional milking of the right udder half at 0430 and 1630 h. Half-udder milk yield and composition were recorded on d 1 and 4, weekly through 35 d in milk (DIM), monthly through 210 DIM, and on d 270±1 of lactation. Udder edema and teat-end scores were evaluated on d 1 and 4, weekly through d 35, and on d 60±1 of lactation. A 1-sided paired t test was used to compare milk yield of 4X- to 2X-udder halves. A 2-sided paired t test was used to test for differences in milk composition, udder edema, and teat-end scores. Overall, 4X stimulated an immediate increase in milk yield. Differential milk yield peaked on d 21 of lactation, with 4X udder halves producing 2.8 kg/d more milk than 2X udder halves (P<0.001). After cessation of 4X, the milk yield differential decreased, but 4X udder halves continued to produce 0.8 kg/d more milk than 2X udder halves through d 270 of lactation (P<0.05). Over the entire lactation, milk component yields and 3.5% fat-corrected milk or energy-corrected milk were greater (P<0.05), whereas somatic cell counts in milk were lower, in 4X udder halves (P<0.05). Udder edema and teat-end condition did not differ between 4X and 2X (P>0.19). In conclusion, IMF during early lactation stimulated a persistent increase in milk yield without negatively affecting several indicators of udder health of primiparous dairy heifers.

Triennial Lactation Symposium: A local affair: How the mammary gland adapts to changes in milking frequency.

Regular removal of milk from the mammary gland is critical to maintaining milk secretion. Early studies in rodents demonstrated that changes in milking frequency influenced mammary blood flow, as well as mammary cell number and activity. Later studies in ruminants confirmed those observations and that the response was regulated locally within the mammary gland. In addition, it was discovered that increased milking frequency (IMF) during early lactation stimulated an increase in milk production that partially persisted through late lactation, indicating long-term effects on mammary function. The local mechanisms regulating the mammary response to IMF are poorly understood, although several have been proposed. To gain insight into the mechanisms underlying the mammary response to IMF, and to identify genes associated with the response, we used a functional genomics approach and conducted experiments on dairy cows exposed to unilateral frequent milking [UFM; twice daily milking (2X) of the left udder half and 4-times daily milking (4X) of the right udder half]. Across multiple experiments, we were unable to detect an effect of UFM on mammary cell proliferation or apoptosis. We have, however, identified distinct transcriptional signatures associated with the mammary response to milk removal and to UFM during early lactation. Sequential sampling of mammary tissue revealed that when UFM was imposed during early lactation, at least 2 sets of genes were coordinately regulated with changes in differential milk production of 4X vs. 2X udder halves. Moreover, some genes were persistently differentially expressed in 4X vs. 2X udder halves after UFM and were associated with the persistent increase in milk yield. We conclude that a coordinated transcriptional response is associated with the increase in milk yield elicited by IMF during early lactation and that the 2 sets of differentially expressed genes may be a marker for the autocrine up-regulation of milk production. Moreover, we propose that we have identified a novel form of imprinting associated with persistent alteration of mammary function, which we term "lactational imprinting."

Acute milk yield response to frequent milking during early lactation is mediated by genes transiently regulated by milk removal.

Milking dairy cows four times daily (4×) instead of twice daily (2×) during early lactation stimulates an increase in milk yield that partly persists through late lactation; however, the mechanisms behind this response are unknown. We hypothesized that the acute mammary response to regular milkings would be transient and would involve different genes from those that may be specifically regulated in response to 4×. Nine multiparous cows were assigned at parturition to unilateral frequent milking (UFM; 2× of the left udder half, 4× of the right udder half). Mammary biopsies were obtained from both rear quarters at 5 days in milk (DIM), immediately after 4× glands had been milked (experiment 1, n = 4 cows), or 2.5 h after both udder halves had last been milked (experiment 2, n = 5 cows). Affymetrix GeneChip Bovine Genome Arrays were used to measure gene expression. We found 855 genes were differentially expressed in mammary tissue between 2× vs. 4× glands of cows in experiment 1 (false discovery rate ≤ 0.05), whereas none were differentially expressed in experiment 2 using the same criterion. We conclude that there is an acute transcriptional response to milk removal, but 4× milking did not elicit differential expression of unique genes. Therefore, there does not appear to be a sustained transcriptional response to 4× milking on day 5 of lactation. Using a differential expression plot of data from both experiments, as well as qRT-PCR, we identified at least two genes (chitinase 3-like-1 and low-density lipoprotein-related protein-2 that may be responsive to both milk removal and to 4× milking. Therefore, the milk yield response to 4× milking may be mediated by genes that are acutely regulated by removal of milk from the mammary gland.

The effects of milk removal or four-times-daily milking on mammary expression of genes involved in the insulin-like growth factor-I axis.

Frequent milking of dairy cows during early lactation elicits both an immediate increase in milk yield and a partial carryover effect that persists to the end of lactation. We hypothesized that the immediate response would be associated with a local increase in insulin-like growth factor (IGF)-I signaling and a consequent increase in mammary growth. Four multiparous cows were assigned at parturition to unilateral frequent milking [UFM; milking of the left udder half twice daily (2x; 0230 and 1430 h); milking of the right udder half 4 times daily (4x; 0230, 0530, 1430, and 1730 h)]. Mammary biopsies were obtained from both udder halves at 5 d in milk at 0530 h (immediately after 4x glands were milked). Incorporation of [3H]-thymidine into DNA and mammary cell apoptosis were not affected by UFM. Because biopsies were obtained when udder halves were at different postmilking intervals, our results reflected both the acute, transient mammary response to milking and the sustained mammary response to frequent milking treatment. We further hypothesized that the acute, transient response involves mechanisms distinct from those regulating the sustained response to frequent milking. To test that hypothesis, mammary biopsies were obtained from UFM cows (n=5) at 0500 h, when time postmilking was the same for both udder halves. Mammary cell apoptosis was not affected by UFM. Expression of genes involved in the IGF-I axis was analyzed to identify acute responses associated with milking, per se, versus sustained responses to frequent milking treatment. Removal of milk from 4x glands was associated with an acute increase in expression of IGF binding protein-1, -3, and -4 mRNA in 2x glands, whereas IGF-I expression was increased by frequent milking treatment. These effects, however, were significant only for expression of IGF binding protein-3. Expression of IGF-I receptor did not differ because of milking frequency but was higher in both udder halves immediately postmilking, indicating a systemic effect. We conclude that several genes of the IGF-I axis respond to milking, per se, or frequent milking treatment, via at least 3 distinct patterns. Increased milking frequency does not alter mammary cell proliferation or apoptosis at 5 d in milk; however, it may increase the bioavailability of IGF-I in the mammary gland. Moreover, the increase in local expression of IGF-I in 4x udder halves indicates a role for this gene in the immediate milk yield response to frequent milking during early lactation.

Use it or lose it: enhancing milk production efficiency by frequent milking of dairy cows.

In the past century, great strides have been made toward optimizing milk production efficiency of dairy cows. One of the key findings that has emerged is that the milk yield of dairy cows is responsive to demands of offspring or milk removal; hence, milk production can be increased by frequent milking. Early studies illustrated the galactopoietic effect of frequent milking during the entire lactation, with 3 times daily milking increasing milk yield by up to 20% relative to twice daily milking. Later studies reported that cows produced more milk during the entire lactation if they were allowed to suckle a calf for the first 3 to 4 mo of lactation. The results of these experiments laid the groundwork for current research, which has identified a time during early lactation wherein the mammary gland of dairy cows is especially receptive to the stimulus of frequent milking. This window of time has been slowly whittled down from the first 10 wk of lactation to the first 6 wk, and it was subsequently established that frequent milking for a short duration within the first 3 wk of lactation can increase milk production through the remainder of lactation [corrected] In addition, there is strong evidence that this milk yield response is locally regulated. Consequently, the concept of "use it or lose it" is becoming more clearly established; that is, the stimulus of frequent milking during early lactation permanently increases the milk production capacity of the mammary gland. Exciting research opportunities now present themselves, and ongoing experiments seek to identify the local factors that are involved in the regulation of milk production efficiency of dairy cows.

Optimal timing and duration of unilateral frequent milking during early lactation of dairy cows.

Increased milking frequency during early lactation can elicit immediate and persistent increases in milk yield; however, the timing and duration of frequent milking have not been optimized. Our objective was to use a half-udder model to determine the milk yield response to 2 wk of frequent milking imposed at 2 different times in early lactation. Multiparous Holstein cows were assigned at parturition to unilateral frequent milking [UFM; twice-daily milking (2x) of the left udder half and 4-times-daily milking (4x) of the right udder half] on d 1 to 14 (UFM-1-14) or 7 to 21 (UFM-7-21) of lactation (n = 10 cows per treatment). Cows were milked 2x before and after UFM. Half-udder milk weights were measured at 1, 3, 7, 14, 21, 28, and 35 d in milk, and then once every 3 mo for the remainder of lactation. For both treatments, the 4x udder halves produced more milk than the 2x udder halves during UFM, resulting in an average difference of 3.7 +/- 0.7 kg/d in UFM-1-14 cows and 2.9 +/- 0.9 kg/d in UFM-7-21 cows. After cessation of UFM, milk production of the 4x udder halves decreased in both treatments, but UFM-7-21 cows produced 1.5 +/- 0.6 kg/d more milk from the 4x side than the 2x side for the remainder of the lactation. In UFM-1-14 cows, the difference was 1.2 +/- 0.7 kg/d, which was significant overall, but not at 270 d in milk. In both treatments, the full-lactation yield of the 4x udder half was greater than the 2x half. Moreover, the total milk yield response to UFM observed in the current study did not differ from that observed in a previous study in which cows were assigned to UFM from d 1 to 21 of lactation. We conclude that UFM for a 2-wk interval during early lactation elicited a persistent increase in milk production of the frequently milked udder half. In addition, the overall milk yield responses observed for UFM-1-14 or UFM-7-21 were not significantly different than that previously observed for UFM on d 1 to 21 of lactation.

The milk yield response to frequent milking in early lactation of dairy cows is locally regulated.

Frequent milking during early lactation of dairy cows increases milk production throughout lactation; however, whether this response is regulated systemically via lactogenic hormones, locally in the mammary gland, or both is unknown. We hypothesized that the effects of frequent milking on milk production during early lactation are regulated via local mechanisms. Ten multiparous cows were assigned at parturition to unilateral frequent milking [UFM; twice daily milking of the left udder half (2x), or 4 times daily milking of the right udder half (4x)] for d 1 to 21 of lactation. After treatment, cows were milked twice daily for the remainder of lactation. At the first milking after calving, milk yield from individual quarters was measured to verify that udder halves produced equal amounts of milk prior to treatment. Thereafter, individual quarters were milked on d 3 and 7, weekly for the first 5 wk of lactation, and once every 3 mo for the remainder of lactation. During UFM, cows produced 3.9 +/- 0.7 kg/d more from the side milked 4x than the side milked 2x. Upon cessation of treatment, milk production from the side milked 4x decreased, but remained at 1.8 +/- 0.5 kg/d more than the side milked 2x for the remainder of lactation. After milk yield was corrected to the equivalent of a whole-udder basis, acute milk yield responses to frequent milking were found to be consistent with previous reports. Moreover, we observed greater persistency in the milk yield response, which lasted throughout lactation. We conclude that both immediate and persistent effects on milk production of frequent milking during early lactation are regulated at the level of the mammary gland. Our results demonstrate that UFM is a valid and efficient model for investigating the effects of frequent milking during early lactation in dairy cows.

Mammary response to exogenous prolactin or frequent milking during early lactation in dairy cows.

Frequent milking of dairy cows during early lactation results in a persistent increase in milk yield; however, the mechanism underlying this effect is unknown. We hypothesized that increased exposure of the mammary gland to prolactin (PRL) mediates the milk yield response. Fifteen multiparous Holstein cows were assigned to 3 treatments for the first 3 wk of lactation: twice daily milking with (2x + PRL) or without (2x) supplemental exogenous PRL, or 4 times daily milking (4x). Mammary biopsies were obtained at 7 DIM, and rates of [(3)H]thymidine incorporation into DNA in vitro were determined. Mammary expression of suppressors of cytokine signaling (SOCS)-1, -2, and -3; the long form of PRL-receptor; and alpha-lactalbumin mRNA was measured by real-time reverse-transcription PCR. Incorporation of [(3)H]thymidine into DNA was not affected by frequent milking or PRL treatment; however, analysis of autoradiograms revealed that stromal cell proliferation was greater in 4x cows. Mammary expression of SOCS-1 was not affected by milking frequency or PRL treatment. Expression of SOCS-2 mRNA was increased with frequent milking or PRL treatment, whereas expression of SOCS-3 mRNA was reduced by frequent milking or exogenous PRL. Abundance of PRL-receptor mRNA was reduced, whereas alpha-lactalbumin mRNA was increased with PRL treatment. These results demonstrate that the bovine mammary gland is responsive to exogenous PRL during early lactation. In addition, differences in the response to frequent milking or exogenous PRL during early lactation indicate distinct effects of PRL and milk removal on the mammary function of dairy cows.